User:Eric Ma/Notebook/iGEM 2010 Lab Prep/2010/05/13: Difference between revisions

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* I will cut Jason's miniprepped DNA because it had the most amount of DNA present.
* I will cut Jason's miniprepped DNA because it had the most amount of DNA present.
* 4 double digests + 4 single cuts, for further confirmation.
* 4 double digests + 4 single cuts, for further confirmation.
* Volumes used:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Control'''
| align="center" style="background:#f0f0f0;"|'''E'''
| align="center" style="background:#f0f0f0;"|'''X'''
| align="center" style="background:#f0f0f0;"|'''S'''
| align="center" style="background:#f0f0f0;"|'''P'''
| align="center" style="background:#f0f0f0;"|'''ES'''
| align="center" style="background:#f0f0f0;"|'''EP'''
| align="center" style="background:#f0f0f0;"|'''XS'''
| align="center" style="background:#f0f0f0;"|'''XP'''
| align="center" style="background:#f0f0f0;"|'''Master Mix'''
|-
| 10X BSA||2||2||2||2||2||2||2||2||2||20
|-
| 10X Buffer 2||2||2||2||2||2||2||2||2||2||20
|-
| EcoRI||0||1||0||0||0||1||1||0||0||
|-
| XbaI||0||0||1||0||0||0||0||1||1||
|-
| SpeI||0||0||0||1||0||1||0||1||0||
|-
| PstI||0||0||0||0||1||0||1||0||1||
|-
| DNA||3||3||3||3||3||3||3||3||3||30
|-
| sdW||30||30||30||30||30||30||30||30||30||300
|-
| Total||37||38||38||38||38||39||39||39||39||370
|}
* Digest for ~1-2 hrs (not so crucial) at room temp.
* Note: Initially wanted only 10 µl of water, but in the end, I calculated for 30 µl instead in the master mix for a total of 300 µl of water. Therefore, the reaction may be a bit dilute, which is why I will let this run while I continue writing and rehearsing the podcast instead.




Revision as of 16:43, 13 May 2010

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Restriction Enzyme Test

  • To test restriction enzymes, digest DNA with E+P, X+S, E+S, X+P; in addition to this, do four single cuts.
  • We can tell which enzyme is not working (if it is such) by examining which pairs are not cutting properly.
  • I will cut Jason's miniprepped DNA because it had the most amount of DNA present.
  • 4 double digests + 4 single cuts, for further confirmation.
  • Volumes used:
Reagent Control E X S P ES EP XS XP Master Mix
10X BSA 2 2 2 2 2 2 2 2 2 20
10X Buffer 2 2 2 2 2 2 2 2 2 2 20
EcoRI 0 1 0 0 0 1 1 0 0
XbaI 0 0 1 0 0 0 0 1 1
SpeI 0 0 0 1 0 1 0 1 0
PstI 0 0 0 0 1 0 1 0 1
DNA 3 3 3 3 3 3 3 3 3 30
sdW 30 30 30 30 30 30 30 30 30 300
Total 37 38 38 38 38 39 39 39 39 370
  • Digest for ~1-2 hrs (not so crucial) at room temp.
  • Note: Initially wanted only 10 µl of water, but in the end, I calculated for 30 µl instead in the master mix for a total of 300 µl of water. Therefore, the reaction may be a bit dilute, which is why I will let this run while I continue writing and rehearsing the podcast instead.



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